Mass Spectrometric Detection of Drug-Resistant HIV

描述（由申请人提供）：美国约有100万人感染HIV-1，每年报告40，000例新病例。虽然这些患者中的大多数从高效抗逆转录病毒疗法（HAART）中获益匪浅，但他们往往会产生与病毒突变相关的多药耐药性，这会给长期治疗带来问题。了解HIV-1逆转录病毒突变谱对于有效规划这些患者的药物治疗极为重要。然而，测序是昂贵的，耗时的，不能检测到低水平的耐药菌株。 本提案的主要目的是开发一种基于无调用蛋白表达和质谱的高灵敏度、高性价比和高通量的HIV-1病毒耐药株检测系统。这种方法是基于这样一个事实，即HIV中的耐药诱导突变改变了编码蛋白质的质量。在I期期间，将使用药物靶向病毒逆转录酶和蛋白酶的野生型（HXB 2）和突变体序列对整体方法进行广泛评价。来自可能含有耐药突变的每种蛋白质区域的肽将在来自兔网织红细胞或E. coli中进行RT-PCR扩增。为了减少无细胞表达多肽的蛋白水解降解，在前期研究中发现了一个问题，即一个高保真的重组翻译系统，该系统基于未修饰的E。将测试大肠杆菌翻译因子。将基于AmberGen的早期工作评价从无细胞提取物中快速分离肽片段的几种方法，包括：i）N-末端表位掺入，ii）tRNA介导的蛋白质工程（TRAMPE）和iii）光裂解生物素。将使用MALDI-TOF质谱法对通过多重PCR和所有下游步骤产生的单个肽和多个肽进行纯化HIV肽的质谱分析。结果将与其他HIV逆转录病毒基因分型方法（包括DNA测序）进行比较。在第二阶段，将根据行业标准96孔或384孔样品格式开发用于HIV-1耐药性检测的高通量MS系统。国际公认的HIV-1耐药性专家Richard D 'Aquila博士将担任该项目的顾问和样本来源。Raymond Tellier博士是一位传染病专家，他使用质谱法来表征丙型肝炎病毒的准种属变异，他也将担任顾问。

DESCRIPTION (provided by applicant): There are approximately one million persons in the U.S. infected with HIV-1, with 40,000 new cases reported each year. While most of these patients greatly benefit from highly active antiretroviral therapy (HAART), they often develop multi-drug resistance associated with viral mutations, which causes problems for long-term treatment. Knowledge of the spectrum of HIV-1 retroviral mutations is extremely important in order to effectively plan drug treatment for these patients. However sequencing is expensive, time-consuming and does not detect low-levels of drug-resistant strains. The principal objective of this proposal is to develop a highly sensitive, cost-effective and high throughput system for detecting drug-resistant strains of HIV-1 virus based on call-free protein expression and mass spectrometry. This approach is based on the fact that drug resistant inducing mutations in HIV alter the mass of the encoded proteins. During Phase I, the overall approach will be extensively evaluated using wild type(HXB2) and mutant sequences of the drug targeted viral reverse transcriptase and protease. Peptides from regions of each protein likely to contain drug-resistant mutations will be expressed in cell-free extracts from rabbit reticulocyte or E. coli using RT-PCR amplified DNA templates. In order to reduce proteolyUc degradation of cell-free expressed peptides, ia problem revealed in preliminary studies, a high fidelity reconstituted translation system based on unmodified E. coli translation factors will be tested. Several methods for rapid peptide fragment isolation from the cell-free extract will be evaluated based on earlier work by AmberGen including: i) N-terminal epitope incorporation, ii) tRNA mediated protein engineering (TRAMPE) and iii) photocleavable biotin. Mass spectrometric analysis of the purified HIV peptides will be performed using MALDI-TOF mass spectrometry both on individual peptides and multiple peptides produced by multiplexing PCR and all downstream steps. Results will be compared to other methods of HIV retrovirus genotyping including DNA sequencing. In Phase II, a high throughput MS based system will be developed for HIV-1 drug-resistance testing based on the industry standard 96- or 384-well sample format. Dr. Richard D'Aquila, an internationally recognized expert on HIV-1 drug-resistance will serve as a consultant on this project and as a source of samples. Dr. Raymond Tellier, an infectious disease expert who has used mass spectrometry to characterize quasi-species variation in Hepatitis C Virus will also serve as a consultant.